Deacetylase genes for the production of phosphinothricin or phosphinothricyl-alanyl-alanine processes for their isolation and their use

ABSTRACT

The invention relates to deacetylase genes, to processes for their isolation, and to their use, in particular for the production of transgenic plants using tissue-specific promoters. It is possible to prevent the development of certain parts in these plants in a targeted manner. With the aid of deacetylase genes, it is furthermore possible to identify and isolate tissue-specific promoters in transgenic plants.

This application is a division of application Ser. No. 08/279,705 , fileJul. 25, 1994, abandoned, which is a continuation of application Ser.No. 08/146,803, filed Nov. 1, 1993, abandoned, which is a continuationof application Ser. No. 07/926,498, filed Aug. 7, 1992, abandoned.

The invention relates to deacetylase genes, to processes for theirisolation, and to their use, in particular for the production oftransgenic plants using tissue-specific promoters. In these plants, thedevelopment of certain parts can be prevented in a targeted fashion.With the aid of deacetylase genes, it is also possible to identify andisolate tissue-specific promoters in transgenic plants.

Phosphinothricin (PTC, 2-amino-4-methylphosphinobutyric acid) is aglutamine synthetase (GS) inhibitor. PTC is a "building block" of theantibiotic phosphinothricyl-alanyl-alanine. This tripeptide (PTT) isactive against Gram-positive and Gram-negative bacteria and also againstthe fungus Botrytis cinerea. PTT is produced by the strain Streptomycesviridochromogenes Tu494 which has been deposited at the DeutscheSammlung fur Mikro-organismen [German Collection of Microorganisms],from where it can be obtained; Deposit Nos. DSM 40736 and DSM 4112.

German Patent 2,717,440 discloses that PTC acts as a total herbicide.The published application (EP-A-0,257,542) describes howherbicide-resistant plants are produced with the aid of aphosphinothricin-N-acetyl-transferase (pat) gene. Thephosphinothricin-N-acetyl-transferase encoded by the pat gene modifiesthe intracellular PTC and detoxifies the herbicide.

The present invention describes deacetylase genes (dea), whoseexpression products are capable of deacetylatingN-acetyl-phosphinothricin (N-Ac-PTC), or N-Ac-PTT, intracellularly,whereupon the antibiotic activity of these compounds is restored.

An N-acetyl-phosphinothricin tripeptide deacetylase gene according tothe invention can be isolated from S. viridochromogenes Tu494. The deagene is located downstream of the pat gene on the 4.0 kb BamHI fragment,which has already been disclosed (EP-A-0,257,542). This gene is locatedon a BgIII-BamHI fragment and is specified in detail by the sequence(FIG. 1 and Table 1). The protein sequence is defined by the DNAsequence. An ATG codon which is recognised in bacteria and in plantsacts as the translation start codon; the Shine-Dalgarno sequence isemphasised by underlining. This gene codes for the last step in PTTbiosynthesis, namely the deacetylation of inactiveN-acetyl-phosphinothricin tripeptide to give the active PTT.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                     GCTCAACTGGCACACCCGCAACGGCGATGTGGAGCCACGCCGGGTGGCCTACGACCGAGC                  CCAGGAGGCCTTCGGGCACCTGGGCCTGCCCCCCGGCGAGACCGTCGTGATCGGCGACTG                  CTCGGCGGAGTGGGTACGGCCCGCCCAGGAGGACGGCAGGACCCTGCTGTACCTGCACGG                  CGGTTCGTACGCCCTCGGATCGCCGCAGTCGCACCGCCATCTGTCCAGCGCGCTGGGCGC                  GGCGGCCGGGGCGGCGGTGCTCGCCCTGCACTACCGCAGGCCGCCCGAGTCTCCCTTCCC                  GGCGGCGGTGGAGGACGCCGTGGCGGCCTACCGGATGCTGCGGGAGCGGGGCCTGCCGCC                  GGGGCGGATCACCTTCGCCGGTGACTCGGCCGGCGCGGGCCTCGCCGTCGCCGCCCTCCA                  GGTGCTGCGCGACGCCGGGGACCCGCTGCCGGCCGCCGCGGTGTGCATCTCGCCCTGGGC                  CGACCTGGCCTGCGAGGGCGCCTCGCACGTCACCCGCAAGGAGCGCGAGATCCTCCTGGA                  CACCGAGGACCTGCTCCGCATGGCGGGGCGCTACCTGGCCGGCACCGATCCCAGGAACCC                  CCTGGCCTCGCCCGCCCACGGCGATCTGACCGGTCTGCCGCCGCTGCTCATCCAGGTCGG                  TTCCGAGGAAGTCCTGTACGACGACGCCCGGGCGCTGGAACAGGCGGCGCTCAAGGCGGG                  CGTACCGGTCACCTTCGACGAGTGGCCGGAGATGTTCCACGTCTGGCACTGGTACCACCC                  GGTGCTCCCCGAGGGGCGTGCCGCCGTCGAGACGGCGGGCGTGTTCCTGCGCCGCGCCACC                  ##STR2##                                                                     __________________________________________________________________________

It is known of many enzymes that their specificity is not limited to onesubstrate. For example, the phosphinothricin-N-acetyl transferase, whichis encoded by the pat gene, is actually used in PTT biosynthesis for theacetylation of desmethyl-PTC and can be used for the detoxification ofPTC due to its non-specificity. Super-expression of the dea gene (withthe aid of suitable promoters or by cloning onto high-copy vectors) itis now possible to use an N-acetyl-PTT-deacetylase of insufficientspecificity for activating N-acetyl-phosphinothricin.

Another dea gene can be obtained from E. coli. In fact, it has beenfound that, in contrast with other bacteria (for example rhizobia andstreptomycetes), no activity can be detected in the so-called PAT assay(Ph.D. thesis Inge Broer, Faculty of Biology, University of Bielefeld,Expression des Phosphinthricin-N-Acetyltransferase-Gens aus Streptomycesviridochromogenes in Nicotiana tabacum [Expression of thephosphinothricin-N-acetyltransferase gene from Streptomycesviridochromogenes in Nicotiana tabacum], p. 42-43, 1989) after cloningthe pat gene into suitable expression vectors (Strauch et al., Gene, 63,65-74, 1988; Wohlleben et al., Gene, 70, 25-37, 1988). Moreover, a lownumber of copies of the pat gene in E. coli is incapable of impartingPTT resistance since the endogenic deacetylase compensates for theaction of the phosphinothricin-N-acetyltransferase. Finally, thisdeacetylase activity can be detected directly by the effectiveinhibition of the GS activity after an addition ofN-acetyl-phosphinothricin. N-Ac-PTC is reacted by the deacetylase togive PTC, which then inhibits the GS in the known manner, which can bemeasured in the γ-glutamyl transferase assay (Bender et al., J.Bacteriol. 129, 1001-1009, 1977). This is due to an endogenicdeacetylase activity of E. coli.

It should be assumed that this activity cannot be found in the argEmutant, which is known from the literature (Baumberg, Molec. Gen.Genetics 106, 162-173, 1970). Other E. coli deacetylase mutants can beselected easily: following traditional (Delic et al., Mut. Res. 9,167-182, 1970; Drake and Baltz, Ann. Rev. Biochem. 45, 11-38, 1976) orTn5 mutagenesis (Kleckner, Ann. Rev. Genet. 15, 341-404, 1981), suchmutants can be recognised on PTT-supplemented minimal medium by the factthat they can only grow after transformation with a pat gene cloned intoa low-copy vector.

Accordingly, the deacetylase gene can be isolated from E. coli byproducing a gene bank, for example in the argE mutant of E. coli, or ina recently isolated mutant, using conventional processes (Maniatis etal., Molecular Cloning: a Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1982).

Methods for isolating further deacetylase genes result from the abovetext: for example isolation of novel organisms which are PTT-sensitivedespite the presence of a pat gene on a low-copy vector, followed byisolation of a deacetylase gene.

In a further aspect of the invention, pat genes and dea genes can beused together with tissue-specific promoters to prevent the developmentof certain plant tissues in a targeted fashion. A specific use is, forexample, the production of male-sterile plants.

The production of hybrid seed in plant breeding depends on theguaranteed avoidance of selfing of the mother plant. In many plantspecies, male-sterile mutants occur naturally, and these are used inbreeding. The molecular mechanism of male sterility (ms) remainsinsufficiently explained as yet. Moreover, no ms variants exist in alarge number of cultivated varieties such as, for example, Betavulgaris. It is therefore of great interest for agriculture to producedefined ms mutants of all important cultured varieties by way ofmolecular genetics. The company PGS/Belgium has proposed such a methodin Patent Application PCT/EP 89/00495. It is based on the destruction ofthe tissue surrounding the pollen mother cells (tapetum). To this end,an RNAase gene is fused with a tapetum-specific promoter (Mariani etal.; Nature 347, 737-741, 1990). The exclusive expression of the gene intapetum cells provides the selective destruction of tissue and thusprevents the formation of mature pollen. A plant carrying this geneshould only be able to produce seeds after cross-fertilization. Anessential shortcoming of this system is the fact that progeny of thisplant are also male-sterile and can therefore not form seeds in thefield, where they depend on selfing. This is only successful when themale partner of the crops carries a gene which can compensate for theaction of the RNAase in the progeny. According to the above-mentioneddisclosed patent application, this is supposed to be effected by thebarstar gene. The fact remains that only genetically modified, i.e.transgenic, partners can be used in the cross.

The text hereinafter proposes methods for the production of ms (malesterility) plants which allow transgenic mother plants to be crossedwith any partners of the same species. This is achieved by combining adea gene which is under the control of, for example, a tapetum promoter,in connection with a constitutively expressed pat gene. Application ofPTC, or PTT, results in a targeted inhibition of the glutaminesynthetase in the tapetum cells, causing their death. An even simplersystem consists in the production of transgenic plants which containonly a single foreign gene, namely a dea gene under the control of atissue-specific promoter, in this case a tapetum promoter, andapplication of N-Ac-PTC, or N-Ac-PTT, to the plant.

Generally speaking, the invention accordingly comprises the followingmethods for the tissue-specific inhibition with the aid of a deacetylasegene, preferably the abovementioned dea gene from E. coli or S.viridochromogenes Tu494:

1) Plants which are resistant to PTT or PTC by pat activity (for exampleproduced as described in EP 0,257,542) are transformed with thedeacetylase gene from streptomycetes under the control of a plant-tissuespecific promoter. Application of PTT or PTC leads to the expression ofthe deacetylase gene for compensating for thephosphinothricin-N-acetyltransferase activity in the respective tissues.These are then destroyed selectively, while the remaining plant isresistant.

2) PTT- or PTC-resistant plants are transformed with the E. colideacetylase gene under the control of a tissue-specific promoter.Application of PTT or PTC leads to the expression of the deacetylasegene for compensating for the phosphinothricin-N-acetyltransferaseactivity in the respective tissues. These are then destroyedselectively, while the remaining plant is resistant.

The use of N-acetyl-phosphinothricin, or N-acetyl-phosphinothricintripeptide, can simplify this system. Both substances are not active asherbicides, but are taken up by plants, translocated and not degradedimmediately. Deacetylase activity for N-acetyl-phosphinothricin andN-acetyl-phosphinothricin tripeptide has not been detected in plants asyet. The above-described 2-gene system can therefore be reduced to a1-gene system and thus greatly simplified as illustrated further below:

3) Any plants are transformed with a deacetylase gene fromstreptomycetes under the control of a tissue-specific promoter. Afterapplication of N-acetyl-phosphinothricin or N-acetyl-phosphinothricintripeptide, the tissue-specific expression leads to the immediate deathof the respective tissue.

4) Any plants are transformed with a deacetylase gene from E. coli underthe control of a tissue-specific promoter. After application ofN-acetyl-phosphinothricin or N-acetyl-phosphinothricin tripeptide, thetissue-specific expression leads to the immediate death of therespective tissue.

Since the specificity of the deacetylase from streptomycetes forN-acetyl-phosphinothricin tripeptide is higher, it will be preferred touse N-acetyl-phosphinothricin tripeptide in case 3) andN-acetyl-phosphinothricin in case 4) if high activities are required.Tissue-specific promoters which can be used are all described promoterswhere selective expression in certain tissues has been detected (forexample Koltunow et al., The Plant Cell., Vol. 2, 1201-1224, 1990). Allnewly-isolated promoters with similar properties are of course, alsosuitable. Other promoters which are suitable in addition totissue-specific promoters are those which are subject to a differenttype of regulation (for example time-dependent, stress-dependent,environment-dependent) and which is tissue-specific.

These methods furthermore allow analysis of the differentiation of cellregulation and the production of plants in which the development ofcertain parts was inhibited in a targeted fashion, preferably theproduction of male-sterile plants.

A further application is the use of a dea gene for the identification ofselectively expressed promoters. If DNA fragments with promoter activityare cloned upstream of dea genes, then the selective disappearance ofparts of tissue after application of N-acetyl-phosphinothricin orN-acetyl-phosphinothricin tripeptide indicates the specificity of thepromoter.

Finally, the invention relates to positive selection systems. Thosecells in which the dea gene has been inactivated can be selected eitherin combination with the pat gene and PTT (or PTC) together with a deagene or with N-acetyl-phosphinothricin (or N-acetyl-phosphinothricintripeptide) and a dea gene alone. This allows successful cloning(insertion inactivation), but also rare events (for exampletransposition), to be selected directly. Other aspects of the inventionare mentioned in the examples.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a restriction map of DNA from cleaving pPRI with BamHI andBgIII; and

FIG. 2 shows restriction maps of vectors used in the invention andcleavage/ligation for preparing vectors of the invention.

EXAMPLE 1

Fusion of the deacetylase encoding region with eucaryotic transcriptionsignals

The plasmid pPRI (see EP-0,257,542) was isolated from an E. coli strainand cleaved with BamHI and BgIII. The digested DNA was separated on anagarose gel, and an 0.9 kb fragment was isolated from the gel. Thevector pROKI (Baulcombe et al., Nature 321, 446-449, 1986) was alsorestricted with BamHI. The two batches were combined and ligated. Theligation mixture was transformed into E. coli S17.1 (Simon et al.,Bio/Technology 1, 784-791, 1983). Colonies growing onkanamycin-containing media were transferred to nitrocellulose filters,incubated for 12 hours at 37° C. and then lysed. The DNA of the bacteriawas fixed on the filter. The 0.9 kb fragment isolated from the agarosegel was made single-stranded by incubation at 100° C. The missing strandwas then synthesized onto the existing strand using Klenow polymeraseand digoxigenin-labeled nucleotides. The labeled strand was used as asample for hybridizing with the bacterial DNA bound to the filter.Hybridizing clones could be detected with the aid of an antibodyreaction. The DNA of the positive clones were isolated by means ofQiagen lysis and digested with BamHI/EcoRI as well as BamHI/HindIII.This restriction allows the orientation of the inserted 0.9 kb fragmentto be determined. The plasmid in orientation I was designated pIB17.1,that of orientation II as pIB17.2 (see FIG. 2).

EXAMPLE 2

Proof of the deacetylation of N-acetyl-PTC and N-acetyl-PTT by thedeacetylase gene

It was possible to demonstrate that the eucaryotic transcription signalscloned in vector pROKI also allow expression in R. meliloti, A.tumefaciens and E. coli.

The plasmids pIB17.1 and pIB17.2 were therefore transferred intoRhizobium meliloti strain 2011 by means of a 2-factorial cross. Byincubation of R. meliloti wild type strains with radiolabeledN-acetyl-PTC, it was possible to demonstrate that this strain does notdeacetylate N-acetyl-PTC. (After incubation of PIB17.1-carrying strainswith N-acetyl-PTC and N-acetyl-PTT, deacetylation can be detected bythin-layer chromatography). It was also possible to demonstrate that R.meliloti reacts highly sensitively to PTC and PTT. Deacetylation cantherefore also be detected via inhibition of the R. meliloti glutaminesynthesase, by the PTC which is liberated.

EXAMPLE 3

Transfer of the modified deacetylase gene into Nicotiana tabacum

The deacetylase gene modified as in Example 1 was transferred into A.tumefaciens LBA4404 by means of a 2-factorial cross. The resultingstrains LBA4404/17.1 and LBA4404/17.2 were used for incubating leafdiscs of Nicotiana tabacum, which were transferred after 3 days to akanamycin-containing shoot induction medium. Regeneratingkanamycin-resistant shoots can be tested for the presence of thedeacetylase gene by Southern hybridization. After treatment withN-acetyl-PTC or N-acetyl-PTT, the plants are then destroyed by the PTC,or PTT, which is liberated.

EXAMPLE 4

Construction of a vector for the transient expression of the modifieddeacetylase gene in E. coli and tobacco protoplasts

The modified deacetylase gene from pIB17.1 and pIB17.2 was cut out ofthe plasmids by digestion with EcoRI/HindIII. The restricted DNA wasseparated in an agarose gel and an 0.9 kb fragment was isolated in eachcase. The vector pSVB28 (Arnold and Puhler, Gene 70, 171-179, 1988) wasalso digested with EcoRI/HindIII. The two batches were combined andligated. After transformation into the β-galactosidase-negative E. colistrain JM83, all clones which carried the vector turned blue, whileclones which carried a vector into which the deacetylase gene had beeninserted remained white. The DNA was isolated from the clones which hadbeen identified in this way and digested with EcoRI/HindIII. The cloneswhich contained the modified deacetylase gene could be recognized on thebasis of the restriction pattern. The vectors which had been constructedare termed pIB27.1 and pIB27.2 (see FIG. 2). They exist in E. coli in alarge number of copies.

EXAMPLE 5

Transient expression of the modified deacetylase gene in tobaccoprotoplasts

The plasmid DNA was isolated from the E. coli strains constructed inExample 4. Young tobacco leaves were incubated with digestion enzymesfor 20 h. The protoplasts which get disengaged from the leaf skeletonwere purified and incubated with polyethylene glycol (PEG) and theisolated DNA in a transfer buffer. The protoplasts were then washed andtaken up in a culture liquid (K3 medium). After incubation for 3 daysunder weak illumination, the regenerating protoplasts were lysed and thecrude extracts were incubated with radiolabeled N-acetyl-PTC andN-acetyl-PTT. The deacetylated PTC or PTT can be detected by thin-layerchromatography.

EXAMPLE 6

Method for the production of male-sterile crop plants using thedeacetylase gene from S. viridochromogenes under the control of atapetum-specific promoter.

The deacetylase gene from Streptomyces viridochromogenes is fused with atapetum-specific promoter from Nicotiana tabacum and introduced intotobacco cells by means of agrobacteria-mediated leaf disctransformation. The plants regenerating from these cells are sprayedwith N-acetyl-PTC or N-acetyl-PTT at any desired point in time beforeanthesis. It can be shown that N-acetyl-PTC is stable in the plant celland transported into all cells. None of the two substances hasnoticeable negative consequences for the wild type plant. As soon as thefirst tapetum cells are formed, they start to express the deacetylasegene. The N-acetyl-PTC or N-acetyl-PTT stored in the cell isdeacetylated by the enzyme and so converted into its active form. Itinhibits the glutamine synthetase of the cells and so results in rapiddestruction. Mature pollen can no longer be formed. In addition, theformation of deacetylase is also interrupted. Cells in the vicinityshould not be affected. If the plant is not treated with N-acetyl-PTC orN-acetyl-PTT, it is fully fertile. This makes compensation for the ms(male sterility) by a gene of the male crossing partner unnecessary. Atthe same time, there exists an accurately defined mutation which has noconsequences on the vitality and usefulness of the plant.

EXAMPLE 7

Identification of tissue-specific promoters in transgenic plants

Tissue-specific promoters can be identified directly in the plant withthe aid of the deacetylase gene from Streptomyces viridochromogenes.

The deacetylase gene is cloned, without a promoter, to the right or leftend of a disarmed T-DNA in such a way that a promoter which is locatedat the insertion site of the T-DNA in the plant genome can read into thegene and so bring about its expression. Transgenic plants are cloned viathe propagation of cuttings. One clone is treated with N-acetyl-PTC orN-acetyl-PTT and examined for tissue which may be in the process ofdying. Using reverse PCR, the gene which has been affected can bemultiplied from a clone which has not been treated with N-acetyl-PTC orN-acetyl-PTT and isolated (Kahl and Weising, Gentransfer bei Pflanzen[Gene Transfer in Plants], Biologie in unserer Zeit, No. 6, p. 181,1988).

EXAMPLE 8

Detection of N-acetyl-phosphinothricin (PPT)-deacetylase activity insoil samples

Soil samples of 500 mg each (sandy loam, Schwanheimer Dune) wereadjusted to 40% of their maximum water capacity and treated with 5 μl ofa 15 mM stock solution of ¹⁴ [C]-L-N-acetyl-PPT. The test samples wereincubated at 28° C. for various periods of time (0 hours, 4, 7, 11 and14 days) and subsequently worked up by extraction with 1×500 μl and1×250 μl of water. 14 μl aliquots from the combined aqueous supernatantswere applied to thin-layer chromatography plates (HPTLC cellulose,Merck) and developed 2×in n-propanol: 25% ammonia=3:2 as the mobilephase. The assays were evaluated by autoradiography. It was possible toidentify N-acetyl-PPT and PPT by comparing the R_(f) values of theradioactive spots with the corresponding reference substances. Itemerged that N-acetyl-PPT in the soil is metabolized within 14 daysalmost completely to give PPT. In contrast, in a control assay withsterile soil samples (soil 4 hours at 200° C.), the substance proved tobe completely stable.

EXAMPLE 9

Isolation and identification of soil microorganisms having anN-acetyl-PPT-specific deacetylase activity 1 g samples of soil wereextracted for 1 hour at room temperature using 10 ml 10 mM NaCl, 10 mMsodium phosphate buffer, pH=7.0. To select various groups ofmicroorganisms, the soil supernatants were plated onto the followingagar media and used for inoculating enrichment cultures in Erlenmeyerflasks with the corresponding liquid media:

(1) MS1 medium (for eubacteria):

5 mM glucose

5 mM succinate

10 mM glycerol

1 g/l NH₄ Cl

50 ml/l solution A

25 ml/l solution B

Solution A:

50 g/l K₂ HPO₄

Solution B:

2.5 g/l MgSO₄

0.5 g/l NaCl

25 ml/l trace elements

(2) Chitin medium (for actinomycetes and streptomycetes as well aschitinovorous bacteria):

10 g/l crab chitin

1 g/l (NH₄)₂ SO₄

0.5 g/l MgSO₄

50 ml/l solution A

1 ml/l trace elements

(3) Antibiotics medium (for higher fungi):

20 g/l malt extract

10 g/l glucose

2 g/l yeast extract

0.5 g/l (NH₄)₂ SO₄

50 μg/ml tetracyclin

All media contained 5 mM N-acetyl-PPT. The agar plates and the liquidcultures were incubated for 3-5 days at 28° C.

20 individual colonies were isolated from each of the selective agarplates and transferred into 5 ml liquid cultures with the correspondingmedium. The cells were allowed to grow for 3-5 days and then centrifugedat 10,000 rpm, and the supernatants were examined in the aminoacidanalyzer (Biotronic LC 5001) for the formation of PPT. In this manner, aPPT-positive culture (CB 10) was isolated from a selection with chitinmedium.

The deacetylase activity of the cells of this culture were subsequentlyadditionally tested by biotransformation with ¹⁴ [C]-L-N-acetyl-PPT asthe substrate. To do this, 1.5 ml of the culture were centrifuged asabove, the cell pellet was washed 1×in 10 mM NaCl, 10 mM sodiumphosphate buffer, pH=7.0, and resuspended in 100 μl of the same buffer.10 μl of the suspension were treated with 10 μl of an 0.25 mM solutionof ¹⁴ [C]-L-N-acetyl-PT and the mixture was incubated for 15 hours at28° C. The bacteria were then centrifuged off, and 7 μl of thesupernatant were analyzed by thin-layer chromatography andautoradiography as described in Example 1. A virtually quantitativereaction of N-acetyl-PPT into PPT could be observed. In addition, theassay showed that the deacetylase found accepts the L enantiomer of theacetylated PPT as substrate.

To further purify the strain with the desired deacetylase activity, theculture CB 10 was plated onto LB agar (10 g/l tryptone, 5 g/l yeastextract, 10 g/l NaCl, 15 g/l agar) and incubated for 2 days at 28° C. 10individual colonies were isolated from the plate, transferred to chitinliquid medium, and the cultures were tested for N-acetyl-PPT deacetylaseactivity as described above. The deacetylase-positive isolates werereplated to check for uniformity of the culture. The strain with thehighest deacetylase activity was identified as Xanthomonas maltophilia(DSM deposit No. DSM 7192).

The enrichment cultures in the soil samples in the various liquid mediawere tested for deacetylation of N-acetyl-PPT as described above. Onlythe chitin medium cultures proved to be deacetylase-positive. Afterthese cultures were plated onto chitin agar, a total of 40 individualcolonies was isolated, grown in chitin liquid medium and subsequentlytested for deacetylase activity. Six positive isolates were Found (BoK1,BoK5, BoK9, BCU1, BCU2, BCU3), from which the active pure cultures wereobtained by replating onto agar plates and culturing further onindividual colonies (see above). The strain with the highest deacetylaseactivity was identified as Microbacterium imperiale (DSM deposit No.7191)

EXAMPLE 10

N-Acetyl-PPT deacetylase enzyme assays with the isolated microorganisms

5 ml precultures of strains BoK1 and BoK5 were grown in LB mediumovernight at 28° C., and 0.5 ml aliquots were transferred to 20 ml of LBmedium or 20 ml of chitin medium containing 1 mM N-acetyl-PPT. The LBcultures were incubated for 15 hours and the chitin cultures for 4 daysin 100 ml Erlenmeyer flasks at 28° C. and 150 rpm. The cells weresubsequently harvested by centrifugation for 10 minutes at 10,000 rpm,the cell pellets were washed 1×in 10 ml mM NaCl, 10 mM sodium phosphatebuffer, pH=7.0, weighed and resuspended in 100 mM tris/HCl, pH=8.0 atc=100 mg/ml. The suspensions were mixed with 1 volume of 100 mMN-acetyl-PPT and incubated in 50 ml Erlenmeyer flasks for 24 hours at28° C. and 220 rpm. The cells were separated by centrifugation for 10minutes at 5000 rpm, and the PPT content in the supernatants was thendetermined in the aminoacid analyzer (see EXAMPLE 9). The results arecompiled in Table 2.

                  TABLE 2                                                         ______________________________________                                        N-Acetyl-PPT deacetylase assays                                               with soil microorganisms                                                                        Concentration                                                                 of PPT in                                                                     the supernatant                                             Strain:     Medium:     [mM]:   [%]*:                                         ______________________________________                                        BoK1        LB          0.7     2.7                                           BoK2        Chitin      13.9    55.5                                          BoK5        LB          6.0     23.9                                          Bok5        Chitin      14.3    57.2                                          ______________________________________                                         *based on the Lenantiomer in the Nacetyl-PPT racemate.                   

EXAMPLE 11

N-Acetyl-PPT deacetylase enzyme assays with actinomycetes

N-Acetyl-PPT-specific deacetylase activities were also found duringfermentation tests with the two actinomycetes strains Actinoplanesliguriae (IFO No. 13997) and Actinoplanes sp. (Strain CollectionZentralforschung No. A 1015) in the presence of N-acetyl-PPT and bybiotransformation with ¹⁴ [C]-L-N-acetyl-PPT as the substrate.

To determine the conversion rates, biotransformations were carried outon the two strains as described in Example 3. The following media wereused:

Medium A:

0.2% yeast extract

0.2% meat extract

0.4% polypeptone (from soya meal)

1% glucose

Medium B:

20 g/l oat flakes

1 ml/l trace elements

The results are compiled in Table 3.

                  TABLE 3                                                         ______________________________________                                        N-Acetyl-PPT deacetylase assays                                               with actinomycetes                                                                                 Concentration                                                                 of PPT in                                                                     the supernatant                                          Strain:       Medium:      [mM]:   [%]*:                                      ______________________________________                                        Actinoplanes liguriae                                                                       A          3.3       13.2                                       (IFO No. 13997)                                                               Actinoplanes liguriae                                                                       B          7.6       30.4                                       (IFO No. 13997)                                                               Actinoplanes sp.                                                                            A          11.0      44.0                                       (No. A 1015)                                                                  Actinoplanes sp.                                                                            B          2.7       10.8                                       (No. A 1015)                                                                  ______________________________________                                         *based on the L enantiomer in the Nacetyl-PPT racemate                   

Further isolates from soil with n-Acetyl-PPT-specific deacetylaseactivity

    ______________________________________                                        from culture CB 10:                                                                          Clavibacter michiganense insidiosum                                           Agrobacterium tumefaciens                                                     Agrobacterium oxydans                                                         Bacillus amyloliquefaciens                                                    Bacillus macerans                                              from culture BoK1:                                                                           Alcaligenes faecalis                                                          Escherichia coli                                               from culture BoK5:                                                                           Staphylococcus hominis                                         from culture BCU1:                                                                           Micrococcus luteus A                                                          Acinetobacter johnsonii                                                       Microbacterium laeraniformans                                  from culture BCU2:                                                                           Acinetobacter calcoaceticus                                    ______________________________________                                    

We claim:
 1. A process for the detecting a tissue-specific specificpromoter, by production of a transgenic plants with selectivelydestroyable tissue, which comprises the following steps:(a) cloning adeacetylase coding region, without a promoter to an end of disarmedT-DNA wherein the deacetylase is capable of deacetylating N-acetyl-PTCor N-acetyl-PTT, (b) transforming plant cells so as to obtain plantcells containing a gene conferring phosphinothricin resistance, and aputative endogenous tissue-specific promoter 5' of end operably linkedto the T-DNA, (c) regenerating from the cells transgenic plants havingtissue portions in which the deacetylase gene is expressed and whereinthe gene conferring phosphinothricin resistance is expressed wherebyphosphinothricin is inactivated and the plants have resistance tophosphinothricin, (d) treating at least one of the transgenic plantswith N-acetyl-PTC or N-acetyl-PTT to cause expression of the deacetylasegene and screening for activity of N-acetyl-PTC or N-acetyl-PTT in thetissue portions such that death of the tissue portions is detected, and(e) ascertaining the deacetylase gene and the tissue-specific promotertherefor effected by the transforming by reverse PCR of a transgenicplant not subject to treatment in step (d), to thereby detect thetissue-specific promoter.
 2. A process as claimed in claim 1 wherein thetissue specific promoter is a pollen- or tapetum-specific promoter andthe death of tissue portions results in male sterility.
 3. A process asclaimed in claim 1 or 2 wherein the deacetylase coding region is an E.coli or S. viridochromogenes deacetylase coding region.